【作者】 林敏；

【导师】 喻树迅；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2014， 博士

【摘要】 棉花是我国重要的经济作物，棉花叶片的早衰严重影响产量和品质。因此，揭示棉花叶片衰老的机制对提高棉花产量和改善纤维品质具有重要的现实和理论意义。本研究以陆地棉中棉所36盛花期的叶片为材料，采用基因组饱和杂交的原理构建了叶片发育全生育期全长均一化cDNA文库。随机选取11,623个克隆进行了测序，获得9,874条高质量的EST序列，经过拼接和组装后得到5,191条Unigene。将这些序列与公共数据库DFCI中已有的棉花EST序列和Unique sequence进行比较分析，结果有2,400条ESTs和991条Unigene是未见报道的。对5,191条Unigene进行功能注释，结果表明这些序列绝大部分与双子叶植物蓖麻（25.0%）、葡萄（23.1%）和杨树（22.2%）中的序列比对上，而与棉花序列注释上的仅有11.2%。该文库为点制芯片和表达谱分析提供了良好的序列基础。进一步利用新一代测序技术对中棉所36成熟叶片和衰老叶片进行了转录组测序，通过序列拼接及聚类处理得到82,867条Unigene，为构建棉花叶片发育和衰老表达谱提供了参考基因序列。随后，根据叶片的表型特征及生化指标（叶绿素和丙二醛含量）的测定，分别构建了六个数字表达谱文库，包括幼嫩期叶片到衰老期叶片。将测序所得的clean tag，采取分级匹配的方式与参考数据库进行匹配，对其中唯一比对到一个基因的Tag（Unambiguous Tags）进行基因注释和分析，结果表明有53%左右的Unambiguous Tags，一共匹配到49,890个基因。我们分别利用转录组测序和荧光定量PCR两种方法验证数字表达谱基因表达量分析的结果，发现这两种方法得到的结果与数字表达谱结果的相关性都比较好，即数字表达谱的结果是可靠的，能用于陆地棉叶片衰老相关基因的表达分析。本实验将构建的数字表达谱进行了分析，鉴定到了4,398个差异表达基因。通过基因表达模式聚类分析，分别得到2,064个上调表达的基因和2,325个下调表达的基因，将差异表达基因进行了pathway显著性富集分析，结果显示一些保护性的途径在上调表达基因中显著富集，而光合作用、卟啉和叶绿素代谢及碳固定等途径在下调基因中显著富集。利用生物信息学方法全面鉴定和分析了差异表达基因中可能参与衰老调控过程的转录因子和激素相关基因及他们的表达模式，本研究鉴定了649个转录因子，其中，MADS，WRKY，C3H，AP2-EREBP家族的基因显著上调表达，鉴定了1,419个参与激素pathway相关的基因，结果表明脱落酸、乙烯、水杨酸、茉莉酸及油菜素内酯可能正调控叶片衰老过程，而细胞分裂素、生长素及赤霉素可能在叶片衰老过程中起到负调控作用。这些相关的基因为研究棉花叶片衰老提供了丰富的候选基因资源和理论依据。同时，本研究从鉴定到的大量叶片衰老相关的候选基因中，用荧光定量PCR实验初步分析了一些参与不同功能的基因的表达模式，并通过克隆和分析GhYLS5/8/9基因，发现它们均在衰老叶片中优势表达，特别是GhYLS9基因，这些基因可以作为研究棉花叶片衰老潜在的分子标记基因。更多还原

【Abstract】 Cotton (Gossypium hirsutum L.) is an important economic crop in China. Leaf prematuresenescence can reduce cotton yield and fiber quality. So it is very important in practice and theory tostydy the mechanism of cotton leaf senescence.In this study, based on the strategy of saturation hybridization with genomic DNA, a normalizedwhole-life-cycle cDNA library was constructed during the plant blooming stage using short seasonupland cotton cultivar CCRI36. The results showed that11,623clones was randomly selected andsequenced,9,874high-quality ESTs were obtained. Thereafter, these ESTs were clustered andassembled into5,191Unigene. Comparative analysis with DFCI database showed that2,400ESTs and991Unigene were new in our library. The functional annotation showed that most Unigene hadsignificant similarity to known genes of dicots, such as Ricinus (25.5%), Vitis (23.1%), Populus (22.2%),whereas only11.2%of the best matches were to cotton. So this library will provide good sequenceresources for preparation chip and gene expression profiling analysis.Two transcriptome libraries were constructed using mature leaf and senescence leaf samples bynext-generation sequencing technology.82,867Unigene were obtained by assembly and clustering. Itwill provide reference sequences for construction of leaf development and senescence expression profile.According to phenotype characteristics and biochemical indices such as chlorophyll andmalondialdehyde levels, six digital gene expression (DGE) libraries were constructed including youngleaf and senescence leaf samples. All clean tags were mapped to the reference sequences, and allowedno more than one nucleotide mismatch, approximately53%of all clean tags could be unambiguouslymapped to one gene in the reference database, and49,890genes were detected. Transcriptome sequencedata and quantitative real-time PCR were performed to demonstrate the utility of DGE profiles. Theresults showed that the data of DGE profiles were reliable for gene expression analysis.In total,4,398genes were identified as differentially expressed genes during leaf senescence.Based on the expression patterns analysis, these genes could be divided into two major groups including2,064up-regulated genes and2,325down-regulated genes. Pathway enrichment analysis revealed thatphotosynthesis, porphyrin and chlorophyll metabolism, metabolic pathways, and carbon fixation weresignificantly overrepresented in the down-regulated pathways, while certain protective activitypathways were significantly enriched in the up-regulated genes.Transcription factor (TF) and hormone related genes that possibly involved in regulating leafsenescence in differentially expressed genes were analyzed by bioinformatics. We identified649TFs,MADS, WRKY, C3H, AP2-EREBP family genes were significantly up-regulated during leafsenescence. Abscisic acid, ethylene, salicylic acid, jasmonic acid and brassinosteroid might havepositive regulation in leaf senescence, whereas, cytokine, auxin and gibberellin might have negativeregulation in the process leaf senescence. These genes provide abundant candidate gene resource andtheoretical basis for clarifying the mechanisms of leaf senescence in cotton. Simultaneously, quantitative real-time PCR were performed to investigate the expression patternsof some representative genes and characterize the role of these genes in the cotton leaf senescence. Thefull length of GhYLS5/8/9genes were cloned and analyzed. The three genes were all up-regulated insenescent leaves, especially GhYLS9. These genes could serve as potential molecular markers fordistinguishing the complex regulatory networks in leaf senescence processes.更多还原